1. Field of the Invention
The present invention relates to protein self-assembly methods and, more particularly, to homo- and hetero-dimeric and multimeric assemblages of proteins and methods for their construction.
2. Description of the Prior Art
Understanding protein-protein interactions is critical to the understanding of how proteins participate in biological regulatory networks. All cellular signaling pathways rely on the joining or breaking of protein interactions to maintain correct function (Fry, D. C., Biopolymers, Peptide Science, 84:535-552, 2006) Protein-interactions have been defined to have different quaternary structures in which the simplest model is dimerization. In the search for new active agents, pharmaceutical companies are targeting therapeutic agents towards the interface of dimerization complexes (Tropsha, A. B. et al., PNAS USA, 88:9488-9492, 1991). In most cases, the goal is to disrupt protein-protein interactions at specific points within a biosignaling pathway (Graddis, T. J. M. et al., Biochemistry, 32:12664-12671, 1993). While knowledge on the subject is substantial and the pharmaceutical industry continues to develop drugs for inhibiting protein-interactions, there still is much to learn about how organisms organize the hundreds of interactions that take place at one time.
Dimerization is a critical structural feature of a myriad of proteins and the regulatory processes in which they participate. While the function of some proteins requires that they be in a dimeric form, the formation may not be directly involved in regulation. Dimerization is, however, involved in signal transduction pathways, repressor and transcription factor operations, enzyme activation and cell-to-cell communication. Dimer formation may occur prior to the signaling or regulatory event or the dimerization itself may be an integral part of the regulatory process.
At physiological concentrations, many dimers are in equilibrium with their monomer components. The dimers are bound together through an interface stabilized by a mixture of hydrophobic interactions, charge attractions and hydrogen bonds. Manipulation of the monomer-dimer equilibrium through ligand binding is a normal part of metabolism. Conformational changes induced by the binding of a ligand is a common way of promoting dimerization and, thereby, regulating the function of the proteins. The complexity of ligands ranges from simple ions to small signal molecules to complex proteins. Metalloregulation, the regulation of processes by the complexation of metal ions to proteins, is common in metal metabolic pathways and some oxidative stress responses. Generally, in metalloregulatory events the binding of metal ions to proteins is thought to induce or stabilize a conformational change in the structure to regulate its activity.
Conventional technology for creating dimers or multimers from proteins that normally are not coupled requires that the proteins be (1) chemically cross-linked, (2) created as direct gene fusions of the proteins involved or (3) indirectly linked by creating gene fusions with specific protein-protein binding motifs so that they, when translated, would be expected to specifically bind to each other through the added motifs with or without the aid of an exogenous regulatory ligand.
Generally, chemical cross-linking requires that the proteins be isolated and either (1) mixed together and treated with reagents to cause them to be covalently attached to each other or (2) each isolated protein be modified with different reagents that will enable the modified proteins to interact when mixed. In the former case, it is difficult to specifically dimerize the proteins into a single or limited series of structures because the crosslinks can form at multiple locations on the surfaces of the two proteins. In the latter case, each protein is modified separately with compounds that will couple with the other when mixed and thereby link the two proteins. Neither of these methods produces entirely specific quaternary structural links between the proteins. Both can lead to conformational changes in the proteins being modified and thus perturb their normal function and in some cases cause the complexes to be poorly soluble. Chemically cross-linked proteins are, however, commonly used in in vivo applications by first isolating the proteins, chemically cross-linking them and then injecting them into an organism. Chemical cross-links frequently are used to link enzymes to antibodies, which subsequently are used analytically in ELISA, tissue fixing or other in vitro analyses.
Chemical cross-linkers capable of diffusing across cell membranes have been used to study the state of oligomerization of dimeric and hexameric species and to probe for heterocomplexes. These studies are best performed on well characterized proteins so that appropriate linkers can be used. The weakness of these methods is that it is not possible to entirely limit the modification to only those proteins being targeted.
Though there are in vitro cleavable chemical cross-linkers, equilibrium between the monomer and dimer cannot be maintained, manipulated or used as a switch. Few of the modifying species can serve as a tool for subsequent affinity purification. Biotinylation, which has been an important tool for in vitro affinity purification (Pierce), cannot be used to purposely construct dimers or higher order species in vivo.
Pierce Biotechnology Inc. markets a wide range of chemical cross-linkers and bioconjugate linkers that interact with a variety of functional groups such as amino, sulphydryls and carboxylates. Some of these cross-linkers diffuse across membranes, but their reactions are largely non-specific and target a broad number of proteins within a cell.
Directly fusing two genes so that they are expressed as one fusion protein is commonly used to add an affinity tag to a protein. The affinity tag then enables the protein fusion to be purified more easily or, in some cases, used as an analytical tool to detect or measure the protein. Proteins, such as glutathione transferase (Pharmacia) and maltose binding protein (NEB); protein domains, such as S-tag (Novagen) and FLAG (Kodak); and 6 histidine repeats (SEQ ID NO: 37) (His tag) frequently are used for affinity purification purposes. Genes or gene fragments also are used to target the fusion protein to specific cellular locations. Novagen (and others) markets a vector that produces a gene fusion of the protein of interest to an export sequence so that the nascent protein will be excreted into the periplasmic space.
The S-tag, FLAG and, especially, the His tag are not as prone to cause folding problems in their fusion partners. However, it is relatively common for the resultant large glutathione transferase and maltose binding protein fusions to have solubility and expression problems. The His tag, which is the most commonly used affinity purification tag on the market today, it the most advantageous affinity tag because it works through a coordination complex and can be used in denaturing conditions. However, although very useful for affinity methods, all of these methods do not enable the construction of homodimers or specific heterospecies.
The direct fusion of the domains derived from the FKDP protein to the proteins of interest can be used to produce homodimers (ARIAD Pharmaceuticals, Inc.) under the control of exogenous rapamycin or related compounds due to the FKDP protein's high affinity for rapamycin. Two FKDP domains bind to each molecule (ligand) or rapamycin. Similarly, using hetero-fusions to FKDP and FRB, domain-specific heterodimers can be induced to form through the addition of a modified rapamycin (ARIAD Pharmaceuticals, Inc.). The regulated feature of these systems is useful and has been used in trafficking studies and may find uses in drug delivery systems. The size and complexity of the fusion proteins, however, pose problems in some cases. The delivery of rapamycin to the cell, tissue or organisms can be difficult. As designed, the monomer to dimer affinity cannot be modified.
There are several cloning systems, so called two-hybrid systems, where the possibility of protein-protein interactions between heteromers can be probed through the formation of dimeric species (Invitrogen and others). The two-hybrid systems also can be used to explore the interactions between specific dimers.
While these systems excel at probing for interactions between one protein and a library of others, they do not enable the formation of homo-dimers of a protein or hetero-dimer formation between proteins selected by the user. The system is, by its nature, linked to a reporter system. In addition, the two-hybrid systems cannot be used to build higher order species, establish an equlibria between the monomer and dimer, etc., or be regulated and used in trafficking studies.
The fusion of genes to a gene or gene fragment, as proposed for the hook motif, enables the formation of homo-dimers in vivo. The possibility of producing fusions of this type has been explored through manipulation of classical leucine-zippers and zinc-fingers. Both of these motifs require a larger sequence be attached to the desired proteins. Both the zipper and finger motifs cannot have their monomer-dimer equilibrium easily manipulated, be used as a switch, be used readily in affinity purification strategies or be used easily to make discrete multimer complexes. To the best of the inventor's knowledge, neither the leucine-zipper nor the zinc finger motifs has been utilized in commercial applications, although the leucine-zipper motif has been explored by an industry group.
With respect to the direct purification of fusion proteins by affinity methods, there are many choices. His-tag (Phanmacia, Sigma and others), S-tag (Novagen), FLAG (Kodak), GST-tag (Phanmacia and others) and maltose binding protein tags all are used in gene fusion based systems to aid in the purification of bacterial and yeast over-expression systems, but these systems are not used in vivo or in vitro to construct dimer or higher order structures and for the most part they are not suited to that task.
There exists a need, therefore, for sequence (“hook”) motifs useful as linkers as well as for a method to link proteins to form specific hetero- and homo-dimeric and multimeric protein structures in the living body in vivo and in a test tube or apparatus in vitro.